We are developing rapid, highly sensitive techniques for mapping sites of phosphorylation in proteins and for following the evolution of complex arrays of phosphorylation/de-phosphorylation events in proteins. Our overall strategy for phosphorylation site mapping is outlined in the steps below: 1) Obtain MALDI-TOF mass spectrum of the intact phosphorylated protein. 2) If feasible, dephosphorylate and obtain MALDI-TOF mass spectrum of the de-phosphorylated protein. 3) Subtract the masses obtained in steps I and 2 to compute the average number of phosphate groups attached to the protein (or at least the number of phosphate-groups released by the phosphotase). 4) If sufficient protein is available, repeat steps 1-3 using ESI-Quadrupole MS. ESI-MS provides a higher resolution measurement but requires more protein and more careful front-end sample preparation and cleanup. We have improved our sensitivity and sample handling by miniaturizing of our desalting columns and optimizing the packing materials used to immobilize the proteins together with careful tuning of the ESI-quadrupole instrument parameters. We can currently obtain high quality mass spectra of certain proteins after loading -I pmol of the protein onto the desalting cartridge. 5)Subject protein to enzymatic or chemical degradation and obtain a MALDI-TOF and MALDI-ITMS map of the resultant proteolysis products. 6)Repeat step 5 with several different degradation reagents to ensure good coverage of the protein. 7) Search the MALDI-TOF spectrum for both unmodified and phosphorylated peptides by comparing the measured masses with those calculated from the known protein sequence and the fragmentation rules of the enzyme. 8) Search the MALDI-ITMS spectrum for all A = 98 Da pairs. We have found that all phosphopeptide ions undergo facile loss of the elements of phosphoric acid in the ion trap and therefore the A = 98 Da pairs provide a convenient signature for phosphopeptides in complex mixture. This loss occurs from phosphoserines, phosphothreonines, and phosphotyrosines. 9) Confirm the phosphorylation site(s) by MALDI-ITMS/MS of the phosphorylated peptide ion - again using the loss of 98 Da as the signature. An alternative to steps 8-9 involves treatment of the peptide mixtures with phosphatase followed by comparison of the treated versus untreated peptide maps. 10) Frequently, the peptide of interest contains multiple candidate sites that potentially may be phosphorylated. To pinpoint the actual site of phosphorylation, w, are testing the practical utility of-(i) LC-Electrospray ionization MS/MS on the triple quadrupole analyzer using constant neutral loss scans and parent of 79- scans (Carr et al). (ii) LC-ESI-MS/MS on our Finnigan LCQ ion trap mass spectrometer making careftil use of the information obtained from the MALDI-TOF-MS experiments. (iii) Ladder sequencing of the peptide of interest